Pulmonary-delivered Anticalin Jagged-1 antagonists reduce experimental airway mucus hyperproduction and obstruction

Abstract

Mucus hypersecretion and mucus obstruction are pathogenic features in many chronic lung diseases directly linked to disease severity, exacerbation, progression, and mortality. The Jagged-1/Notch pathway is a promising therapeutic target that regulates secretory and ciliated cell trans-differentiation in the lung. However, the Notch pathway is also required in various other organs. Hence, pulmonary delivery of therapeutic agents is a promising approach to target this pathway while minimizing systemic exposure. Using Anticalin technology, Jagged-1 Anticalin binding proteins were generated and engineered to potent and selective inhalable Jagged-1 antagonists. Their therapeutic potential to reduce airway mucus hyperproduction and obstruction was investigated ex vivo and in vivo. In primary airway cell cultures grown at an air-liquid interface and stimulated with inflammatory cytokines, Jagged-1 Anticalin binding proteins reduced both mucin gene expression and mucous cell metaplasia. In vivo, prophylactic and therapeutic treatment with a pulmonary-delivered Jagged-1 Anticalin binding protein reduced mucous cell metaplasia, epithelial thickening, and airway mucus hyperproduction in IL-13 and house dust mite allergen-challenged mice, respectively. Furthermore, in a transgenic mouse model with pathophysiologic features of cystic fibrosis and chronic obstructive pulmonary disease (COPD), pulmonary-delivered Jagged-1 Anticalin binding protein reduced hallmarks of airway mucus obstruction. In all in vivo models, a reduction of mucous cells with a concomitant increase of ciliated cells was observed. Collectively, these findings support Jagged-1 antagonists' therapeutic potential for patients with muco-obstructive lung diseases and the feasibility of targeting the Jagged-1/Notch pathway by inhalation.NEW & NOTEWORTHY Airway mucus drives severity and mortality in diverse chronic lung diseases. The Jagged-1/Notch pathway controls the balance of ciliated versus mucous cells, but targeting the pathway systemically carries the risk of side effects. Here we developed novel, Anticalin-derived, pulmonary-delivered Jagged-1 antagonists, to inhibit airway mucus hyperproduction and obstruction in chronic lung diseases. Our preclinical data demonstrate the effectiveness of these antagonists in diminishing secretory cell and mucus levels and alleviating hallmarks of mucus obstruction.

Keywords: Jagged-1/Notch; biologic; inhaled; mucus; secretory cells.

Graphical abstract

Figure 1.

Jagged-1 Anticalin binding proteins 1 and 2 dose-dependently inhibit Jagged-1/Notch2 signaling in vitro and selectively bind to Jagged-1. A and B: dose-dependent inhibition of Jagged-1/Notch2 signaling by Jagged-1 Anticalin binding protein (JAc) 1 and 2 was assessed in a cell-based reporter assay in vitro. Human T-Rex-293 cells expressing human (hu) or murine (mu) Jagged-1 via doxycycline-induction were co-cultured with a reporter cell line endogenously expressing hu (A) or mu (B) Notch2, respectively. The binding of Jagged-1 to Notch2 leads to the activation of the Notch signaling pathway and the expression of the luciferase reporter gene. The Luciferase signal was measured as a relative light unit and normalized to the baseline signal. C and D: binding of JAc1 (C) and JAc2 (D) to recombinant hu or muJagged-1 and other huNotch ligand proteins was assessed in a Luminex MagPlex bead assay. A and B depict one representative experiment with one biological replicate and three technical replicates, data shown as means ± SD, and C and D depict one representative experiment with one biological replicate and one technical replicate. MFI, mean fluorescence intensity.

Figure 2.

Mucous cell metaplasia and mucus production in IL-13 or IL-17-stimulated air-liquid interface cell cultures were reduced by Jagged-1 Anticalin binding protein treatment. A: experimental design. Air-liquid interface (ALI)-cultured human bronchial epithelial cells (HBECs) were stimulated with 1 ng/mL recombinant human (hu) IL-13 (B and D) or 10 ng/mL recombinant huIL-17A (C and E) every other day in conjunction with Jagged-1 Anticalin binding protein (JAc) 1, JAc2 or lipocalin isotype control protein (Control Ac) at concentrations of 20 nM (B and D) or 100 nM (C and E). B and C: gene expression values of MUC5AC, MUC5B, and FOXA3 were assessed by RT-qPCR, relative to HPRT, for huIL-13- (B) and huIL-17A-stimulated (C) ALI cell cultures after 7 days. D and E: representative AB-PAS stainings of vertical ALI HBEC culture sections stimulated with huIL-13 (D) or huIL-17A treatment (E) were performed to visualize mucus content after 10 days of treatment. Scale bar: 100 µm. Data represent a biological N = 1 of one experiment performed with HBECs from one donor. Graphs show values of three technical replicates with means ± SD. Statistical significance was assessed by the nonparametric Kruskal–Wallis test followed by Dunn’s multiple comparison test. *P ≤ 0.05, ns, not significant. AB-PAS, Alcian blue-periodic acid-Schiff; HBECs, human bronchial epithelial cells.

Figure 3.

Anticalin proteins penetrate mucus and airway surface liquid layer in air-liquid interface cell cultures from cystic fibrosis-derived donors and show no interaction with mucus. A: mucus and airway surface liquid layer (ASL) penetration of Jagged-1 Anticalin binding proteins (JAcs) was assessed by confocal microscopy in cystic fibrosis (CF)-derived and air-liquid interface (ALI) cultured primary human bronchial epithelial cells. ALI cell cultures were left undisturbed for 5 days to generate a mucus-rich ASL and were then stained with 10-kDa TAMRA-dextran (ASL, red) and 25-nm FITC microspheres (mucus, green) 24 h before the experiment. Fluorescent labeled JAcs, or mucus binding microspheres (blue), were added apically to the ALI cell cultures at t = 0, and cross-sectional (XZ orientation) images were captured after 0.5 min or 5 min, respectively. Overlay of ASL and JAcs or control beads appears in purple (top and bottom right channel). Bar size: 10 µm. B: quartz crystal microbalance with dissipation (QCM-D) was used to analyze the mucus/mucin interaction with JAcs (Drug) by measuring the frequency shift (blue, changes in mucus mass) and dissipation shift (red, changes in mucus hydration) and a positively charged lysine-rich control peptide (PLL) that interacts with the semi-purified, native mucus preparation. Images and graphs are representative of n = 3 separate experiments and of three tested precursor JAcs exhibiting different isoelectric points of 5, 5.1, and 7.8. The term “Treatment” marked in blue means either the addition of JAcs (top) or control beads (bottom) as indicated in A.

Figure 4.

Jagged-1 Anticalin binding protein 2 treatment dose-dependently prevents IL-13-induced mucous cell metaplasia and mucus hyperproduction in vivo. A: experimental design. Murine recombinant IL-13 (muIL-13; 10 µg/mouse) or saline was administered by oropharyngeal aspiration (o.a.) to mice once a day for 3 days (D0–D2), as indicated by blue arrows. Jagged-1 Anticalin binding protein (JAc) 2 at doses of 0.05, 0.1, 0.5, and 1 mg/kg, or control vehicle, was intratracheally (i.t.) instilled 2 h prior to muIL-13 at D0–D2, as indicated by brown arrows. Mice were euthanized at D3. B: RT-qPCR analysis of Muc5ac and Muc5b, relative to Hprt. C and D: mucus score and number of mucus secretory cells (D) were quantified based on PAS stainings of histological tissue sections, with representative images shown in (C). E and F: FOXJ1 immunolabeling was performed to quantify ciliated cells (per 150 µm basal lamina length) (F); representative images are shown in (E). G: RT-qPCR of Foxj1, relative to Hprt. Dots represent 7–10 biological replicates. Data are shown as means ± SD. Statistical significance was assessed by the nonparametric Kruskal–Wallis test followed by Dunn’s multiple comparison test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, and ns, not significant; A.U., arbitrary units; scale bar: 100 µm. Enlarged image inserts: zoom factor 4. PAS, periodic acid-Schiff.

Figure 5.

Jagged-1 Anticalin binding protein 2 treatment reduces IL-13-induced mucous cell metaplasia and mucus hyperproduction in vivo. A: experimental design. Murine recombinant IL-13 (muIL-13) or saline was administered by oropharyngeal aspiration (o.a.) to mice once a day for 10 days (D0–D2: 10 µg/mouse; D3–D9: 1 µg/mouse), as indicated by blue arrows. Jagged-1 Anticalin binding protein (JAc) 2 at doses of 0.1 and 1 mg/kg, or control vehicle, was administered by o.a. daily 2 h prior to muIL-13 on D3–D9 as indicated by brown arrows. Mice were euthanized at D10. B and C: PAS stainings (B, top) and FOXJ1 immunolabeling (B, bottom) of lung tissue sections for each treatment group were performed to quantify changes in mucus secretory cell numbers (C), mucus score (D), epithelial height (E), and ciliated (FOXJ1⁺) cell numbers per 150 µm basal lamina length (F). Dots represent 6–10 biological replicates. Data are shown as means ± SD. Statistical significance was assessed by the nonparametric Kruskal–Wallis test followed by Dunn’s multiple comparison test. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001, ns, not significant; A.U., arbitrary units; scale bar: 100 µm; enlarged image inserts: zoom factor 4. PAS, periodic acid-Schiff.

Figure 6.

Jagged-1 Anticalin binding protein 2 treatment reduces house dust mite-induced mucous cell metaplasia and mucus hyperproduction in vivo. A: experimental design. House dust mite (HDM) (100 µg/mouse) allergen or saline was administered to mice once a week (D0–D41) by oropharyngeal aspiration (o.a.), as indicated by blue arrows. Jagged-1 Anticalin binding protein (JAc) 2 (1 mg/kg) or control vehicle was administered by o.a. daily from D15 to D41, as indicated by brown arrows. Mice were euthanized on D15 (termination I) or on D42 (termination II). B–F show data from termination II. B: RT-qPCR analysis of Muc5ac and Muc5b expression, relative to Hprt. C–E: mucus score and number of mucus secretory cells (D) and epithelial height (E) were quantified based on PAS stainings of histological tissue sections, with representative images shown in C. F and G: representative images of FOXJ1 immunolabeling (F) and quantification of FOXJ1⁺ cells per 150 µm basal lamina length (G). Dots represent 8–10 biological replicates. Data are shown as means ± SD. Statistical significance was assessed by the nonparametric Kruskal–Wallis test followed by Dunn’s multiple comparison test. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001, ns, not significant; A.U., arbitrary units; scale bar: 100 µm; enlarged image inserts: zoom factor 4; Q.D., once a day; PAS, periodic acid-Schiff.

Figure 7.

Jagged-1 Anticalin binding protein 2 treatment reverses airway mucus obstruction in Scnn1b-Tg mice. A: experimental design. Jagged-1 Anticalin binding protein (JAc) 2 (5 mg/kg) or control vehicle were administered by oropharyngeal aspiration (o.a.) to Scnn1b-Tg mice daily from postnatal day (PND) 22 to 28, as indicated by brown arrows. Mice were euthanized on D29. B: RT-qPCR analysis of Muc5ac and Muc5b, relative to Tbp. C–E: morphometric analysis of luminal (D) and epithelial (E) airway mucus volume density (Vs) in PND29 mice quantified based on AB-PAS staining of proximal lobes, with representative images shown in C. F and G: representative images of FOXJ1 immunolabeling (F) and quantification of FOXJ1⁺ cells per 150 µm basal lamina length (G) in proximal lung sections. Dots represent 7–12 (B), 9–13 (D and E), or 8–11 (G) biological replicates. Data are shown as means ± SD. Statistical significance was assessed by the nonparametric Kruskal–Wallis test followed by Dunn’s multiple comparison test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ns, not significant; scale bar: 100 µm. AB-PAS, Alcian blue-periodic acid-Schiff.